DESCRIPTION: Vascular leak is a hallmark of tissue injury, and results in the movement of plasma into the interstitium where immune mediators and clotting factors invade the injured tissue. Left to persist, vascular leak can exacerbate inflammation and lead to a chronic and abnormal wound healing response. Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator that circulates in the bloodstream where it tonically enhances vascular barrier integrity. S1P is synthesized by sphingosine kinase, which exists as two isotypes (SphK1 and SphK2). Recently, SphynKx Therapeutics discovered an excellent lead SphK2 selective inhibitor that has been validated to inhibit the enzyme in vivo using changes in circulating S1P level as the biomarker of target engagement. Perhaps counterintuitive, this lead SphK2 inhibitor causes circulating S1P levels to nearly double when given to mice with both SphK1 and SphK2 alleles (i.e., wild type mice). This result might seem odd until one considers that blood S1P levels in SphK2 null mice are 2-3 times higher than in wild type littermates (and about 6-fold higher than in SphK1 null littermates). Given this finding, we hypothesize that raising blood S1P levels through SphK2 inhibition will improve endothelial barrier function and halt or slow the progression of diseases with a significant leak component. More specifically, we are focused on exploring the therapeutic utility of our SphK2 inhibitors to treat tissue fibrosis, where vascular leak results in a constant influx of pro-fibrotic mediators that cause abnormal wound healing. Although promising, SphynKx's lead SphK2 inhibitor must be optimized further to advance this molecule into further proof-of-concept testing and conduct investigational new drug (IND)-enabling studies. In particular, this project seeks to improve the potency and isotype selectivity of this compound towards SphK2 over SphK1. Therefore, the goal of our project is (aim 1) to optimize the structure of the lead compound using medicinal chemistry, (aim 2) characterize candidate compounds using an algorithm that includes in vitro target screens and in vivo pharmacokinetics, and finally (aim 3) evaluate the ability of select compounds to protect against vascular leak and attenuate fibrosis in two mouse models of kidney fibrosis. If we are successful, our investigations will have validated SphK2 as a drug target in vascular leak and fibrosis, and delivered an optimized lead SphK2 inhibitor for further pre-clinical development.